Cytotoxic compounds

ABSTRACT

Compounds are described that bind to tubulin causing tubulin depolymerization and inhibiting tubulin polymerization. The compounds of the invention are therapeutically effective to inhibit cellular proliferation, for example, as effective anti-cancer agents. The compounds can also induce cytotoxicity in cells such as leukemia cells. The chemical structure of the compounds includes a furan, thiophene, thiazole, oxazole, or imidazole group at one end of the molecule (head) and a hydrophobic, aliphatic chain at the other end of the molecule (tail).

PRIORITY OF THE APPLICATION

[0001] This application claims priority to U.S. Provisional Application60/285,215 filed Apr. 20, 2001.

FIELD OF THE INVENTION

[0002] The invention relates to novel compounds that are effectiveanti-cancer agents and potent inhibitors of tubulin polymerization.

BACKGROUND OF THE INVENTION

[0003] Cancer is a major disease that continues as one of the leadingcauses of death at any age. In the United States alone, it is estimatedthat more than a half a million Americans will die annually of cancer.Currently, radiotherapy and chemotherapy are two important methods usedin the treatment of cancer.

[0004] Considerable efforts are underway to develop new chemotherapeuticagents for more potent and specific anti-cancer therapy, presentingeffective and efficient cytotoxicity against tumor cells, with minimalinterference with normal cell function. Accordingly, there is an urgentneed for the development and analysis of novel, effective anti-canceragents.

[0005] Cellular proliferation, for example, in cancer, occurs as aresult of cell division, or mitosis. Microtubules play a pivotal role inmitotic spindle assembly and cell division. These cytoskeletal elementsare formed by the self-association of the αβ tubulin heterodimers.

[0006] Recently, the structure of the αβ tubulin dimer was resolved byelectron crystallography of zinc-induced tubulin sheets. According tothe reported atomic model, each 46×40×65 Å tubulin monomer is made up ofa 205 amino acid N-terminal GTP/GDP binding domain with a Rossman foldtopology typical for nucleotide-binding proteins, a 180 amino acidintermediate domain comprised of a mixed β sheet and five helices whichcontain the taxol binding site, and a predominantly helical C-terminaldomain implicated in binding of microtubule-associated protein (MAP) andmotor proteins.

[0007] As disclosed in U.S. Pat. No. 6,258,841 B1, tubulin has a bindingpocket in a region within the intermediate domain of tubulin, locatedbetween the GDP/GTP binding site and the taxol binding site. Theapproximate dimensions of the binding pocket are 6 Å×22 Å×7 Å. Thepocket, referred to as the COBRA binding pocket, has an abundance ofleucine residues (7 leucine and 2 isoleucine) providing a highlyhydrophobic binding environment. It is characterized by a narrow cavitywith elongated dimensions, suitable for accommodating an aliphatic chainof up to about 12 carbons.

[0008] Compounds that bind with the COBRA binding pocket can interferewith tubulin polymerization and can provide novel agents for thetreatment of cancer.

SUMMARY OF THE INVENTION

[0009] The compounds of the invention bind to tubulin causing tubulindepolymerization and inhibiting tubulin polymerization. The tubulinbinding compounds of the invention are therapeutically effective toinhibit cellular proliferation, for example, as effective anti-canceragents. The compounds are cytotoxic against tumor cells such as leukemiacells. The compounds are novel furan, thiophene, thiazole, oxazole, orimidazole derivatives.

[0010] One aspect of the invention includes COBRA compounds representedby the general formula I:

[0011] where

[0012] X is O, S, or NH;

[0013] R is a saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain;

[0014] R¹ is hydrogen, halogen, OH, (C₁ to C₆) alkoxy, (C₁ to C₆) acyl,(C₁ to C₆) ester, or (C₁ to C₆) carboxylic acid;

[0015] Y is OH, SH, CN, halogen, or (C₁ to C₆) alkoxy; and

[0016] pharmaceutically acceptable salts thereof.

[0017] In another aspect of the invention, COBRA compounds are providedof the general formula II:

[0018] where

[0019] X is NH, O, or S;

[0020] R is a saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain;

[0021] R² is hydrogen, OH, (C₁ to C₆) alkoxy, (C₁ to C₆) alkyl, (C₁ toC₆)alkenyl, (C₁ to C₆) alkynyl, (C₃ to C₇) cycloalkyl, aryl, heteroaryl,halogen, (C₁ to C₆) acyl, (C₁ to C₆) ester, or (C₁ to C₆) carboxylicacid;

[0022] Y is OH, SH, CN, halogen, or (C₁ to C₆) alkoxy; and

[0023] pharmaceutically acceptable salts thereof.

[0024] In yet another aspect of the invention, the COBRA compound hasthe general formula III:

[0025] where

[0026] X is NH, O, or S;

[0027] R is a saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain;

[0028] R² is hydrogen, OH, (C₁ to C₆) alkoxy, (C₁ to C₆) alkyl, (C₁ toC₆)alkenyl, (C₁ to C₆) alkynyl, (C₃ to C₇) cycloalkyl, aryl, heteroaryl,halogen, (C₁ to C₆) acyl, (C₁ to C₆) ester, or (C₁ to C₆) carboxylicacid;

[0029] Y is OH, SH, CN, halo, or (C₁ to C₆) alkoxy;

[0030] and pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0031] All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

[0032] As used herein, “acyl” refers to a group containing a carbonattached to oxygen by a double bond. Acyl groups can be a part of, forexample, aldehydes or ketones. Typically, acyl groups include 1 to 15carbon atoms or 1 to 6 carbon atoms. The carbon atoms can be aliphaticor aromatic. In some embodiments, the acyl group has 1 to 3 carbon atomsor 1 carbon atom.

[0033] The term “alkyl” refers to straight or branched hydrocarbonradicals, such as methyl, ethyl, propyl, butyl, pentyl, octyl,isopropyl, tert-butyl, sec-butyl, and the like. Typically, alkyl groupsinclude 1 to 15 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbonatoms.

[0034] As used herein, “alkythio” comprises a sulfur attached to analkyl by a single bond. Typically, alkythio groups include 1 to 15carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms.

[0035] As used herein, “alkene” and “alkenyl”, includes both branchedand straight chain aliphatic hydrocarbon groups that have at least onedouble bond. Typically, alkene groups include 1 to 15 carbon atoms, 1 to6 carbon atoms, or 1 to 3 carbon atoms.

[0036] The term “alkoxy” refers to an oxygen atom substituted with analkyl radical as defined above. Typical alkoxy groups include 1 to 15 or1 to 6 carbon atoms or 1 to 3 carbon atoms such as methoxy, ethoxy,propoxy, iso-propoxy, and the like. Preferable alkoxy groups includemethoxy and ethoxy.

[0037] As used herein, “alkyne” and “alkynyl” includes both branched andstraight chain aliphatic hydrocarbon groups that have at least onetriple bond. Typically, alkyne groups include 1 to 15 carbon atoms, 1 to6 carbon atoms, or 1 to 3 carbon atoms.

[0038] As used herein “amine” refers to compounds of the formulaNR^(a)R^(b) and includes primary, secondary, and tertiary amines. R^(a)and R^(b) are each independently hydrogen, (C₁ to C₆) alkyl, aryl,heteroaryl, (C₁ to C₆) acyl, (C₃ to C₇) cycloalkyl, or R^(a) and R^(b)taken together with the nitrogen to which they are attached form a ringsuch as pyrrolidino, piperidino, morpholino, or thiomorpholino.

[0039] The term “aryl” refers to monovalent unsaturated aromaticcarbocyclic radicals having a single ring, such as phenyl, or multiplefused rings, such as naphthyl or anthryl.

[0040] As used herein, “aryloxy” comprises an oxygen attached to an arylby a single bond.

[0041] As used herein, “cycloalkyl” includes cyclic alkanes having 3 to7 carbon atoms.

[0042] As used herein, an “ester” comprises a carbon attached to a firstoxygen by a double bond and to a second oxygen by a single bond. Thesecond oxygen is also attached to an alkyl group which has 1 to 15carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms.

[0043] As used herein, the term “carboxylic acid” comprises a carbonattached to a first oxygen by a double bond and to a second oxygen by asingle bond. The second oxygen is also attached a hydrogen atom, i.e.COOH.

[0044] As used herein, the terms “halogen” or “halo” refers to fluoride,chloride, bromide, and iodide radicals.

[0045] As used herein, “heteroaryl” includes substituted orunsubstituted aromatic hydrocarbon compounds having at least one atom ofO, N or S in an aromatic ring. Typical heteroaryl groups include, forexample, furan, thiophene, pyrrole, thiazole, oxazole, or imidazolegroup. Heteroaryl groups can include two aromatic groups fused togethersuch as, for example, benzothiophene, indole, carbazole, quinazoline,quinoline, and purine.

[0046] As used herein, the term “thioacyl” refers to a group comprisinga carbon atom attached to a sulfur atom by a double bond. Typically, thethioacyl group has 1 to 6 carbon atoms or 1 to 3 carbon atoms.

[0047] As used herein, “pharmaceutically acceptable salt thereof”includes an acid addition salt or a base salt.

[0048] As used herein, ” pharmaceutically acceptable carrier” includesany material which, when combined with a compound of the invention,allows the compound to retain biological activity, such as the abilityto induce apoptosis of leukemia or breast tumor cells, and isnon-reactive with the subject's immune system. Examples include, but arenot limited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsions, and various types of wetting agents. Compositions comprisingsuch carriers are formulated by well known conventional methods (see,for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed.,Mack Publishing Co., Easton, Pa.).

[0049] “Treating” or “Treatment” in the context of this invention meansthe prevention or reduction in severity of symptoms or effects of apathological condition, including prolonging life expectancy. In thecontext of cancer therapy, treatment includes prevention of tumorgrowth, reduction of tumor size, enhanced tumor cell death, andincreased apoptosis.

COBRA Binding Pocket on Tubulin

[0050] The COBRA binding pocket accommodates COBRA compounds. As usedherein, the phrase “COBRA compound” refers to a compound having anon-linear polar group at one end of a molecule (the head) and aaliphatic, hydrophobic group at the other end of the molecule (thetail). The structure of the COBRA compounds increases the attraction ofthe compounds to the COBRA pocket and increases the residence time ofthe molecule in the binding pocket.

[0051] The residues of the COBRA binding pocket suitable for interactionwith the tail part of COBRA compounds include Asp367, Leu217, Val275,Ile 276, Leu368, Tyr272, Ile212, Ile234, Gln233, Leu230, His229, Ile209,Ile231, and Leu23. Residues of the pocket suitable for interaction withthe head part of COBRA compounds include Asn226, Pro222, and Ile219.

Compounds of the Invention

[0052] In general, the COBRA compounds of the invention include analiphatic, hydrophobic tail part and a non-linear polar head part. Thehead part is a furan, thiophene, pyrrole, thiazole, oxazole, orimidazole group. The compounds are suitable for binding to the COBRAbinding pocket of tubulin.

[0053] One aspect of the invention includes COBRA compounds representedby the general formula I:

[0054] where

[0055] X is O, S,or NH;

[0056] R is a saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain;

[0057] R¹ is hydrogen, halogen, OH, (C₁ to C₆) alkoxy, (C₁ to C₆) acyl,(C₁ to C₆) ester, or (C₁ to C₆) carboxylic acid;

[0058] Y is OH, SH, CN, halogen, or (C₁ to C₆) alkoxy; and

[0059] pharmaceutically acceptable salts thereof.

[0060] The saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain can bean alkyl, an alkenyl, or an alkynyl group. In some embodiments, thegroup is a C₁₂ alkyl or a C₁₂ alkylene. In one embodiment, R is C₁₂H₂₅.

[0061] The acyl, ester, carboxylic acid, or alkoxy groups can besubstituted or unsubstituted. Suitable substituents include, forexample, halogens, hydroxyl, amino, amino alkyl, acyl, thioacyl, CN, SH,ester, thioester, alkoxy, aryloxy, and alkylthio.

[0062] In some embodiments, R is C₁₂H₂₅; R¹ is hydrogen, bromine,chlorine, CHO or COOH; Y is OH; and X is oxygen or sulfur. For example,the COBRA compounds of formula I can be:

[0063] In another aspect of the invention, COBRA compounds are providedof the general formula II:

[0064] where

[0065] X is NH, O, or S;

[0066] R is a saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain;

[0067] R² is hydrogen, OH, (C₁ to C₆) alkoxy, (C₁ to C₆) alkyl, (C₁ toC₆)alkenyl, (C₁ to C₆) alkynyl, (C₃ to C₇) cycloalkyl, aryl, heteroaryl,halogen, (C₁ to C₆) acyl, (C₁ to C₆) ester, or (C₁ to C₆) carboxylicacid;

[0068] Y is OH, SH, CN, halogen, or (C₁ to C₆) alkoxy; and

[0069] pharmaceutically acceptable salt thereof.

[0070] The saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain can bean alkyl, an alkenyl, or an alkynyl group. In some embodiments, thegroup is a C₁₂ alkyl or a C₁₂ alkylene. In one embodiment, R is C₁₂H₂₅.

[0071] The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl,ester, carboxylic acid, and alkoxy groups can be substituted orunsubstituted. Suitable substituents include, for example, halogens,hydroxyl, amino, amino alkyl, acyl, thioacyl, CN, SH, ester, thioester,alkoxy, aryloxy, and alkylthio.

[0072] In some embodiments of formula II, R is C₁₂H₂₅; Y is OH; X is Sor NH; and R² is hydrogen or a (C₁ to C₆) alkyl. For example, the COBRAcompounds of formula II include

[0073] In yet another aspect of the invention, the COBRA compound hasthe general formula III:

[0074] where

[0075] X is NH, O, or S;

[0076] R is a saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain;

[0077] R² is hydrogen, OH, (C₁ to C₆) alkoxy, (C₁ to C₆) alkyl, (C₁ toC₆)alkenyl, (C₁ to C₆) alkynyl, (C₃ to C₇) cycloalkyl, aryl, heteroaryl,halogen, (C₁ to C₆) acyl, (C₁ to C₆) ester, or (C₁ to C₆) carboxylicacid;

[0078] Y is OH, SH, CN, halo, or (C₁ to C₆) alkoxy; and

[0079] pharmaceutically acceptable salt thereof.

[0080] The saturated or unsaturated (C₇ to C₁₅) hydrocarbon chain can bean alkyl, an alkenyl, or an alkynyl group. In some embodiments, thegroup is a C₁₂ alkyl or a C₁₂ alkylene. In one embodiment, R is C₁₂H₂₅.

[0081] The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, acyl,ester, carboxylic acid, and alkoxy groups can be substituted orunsubstituted. Suitable substituents include, for example, halogens,hydroxyl, amino, amino alkyl, acyl, thioacyl, CN, SH, ester, thioester,alkoxy, aryloxy, and alkylthio.

[0082] In some embodiments of formula III, R is C₁₂H₂₅; Y is OH; X is Sor NH; and R² is hydrogen or a (C₁ to C₆) alkyl. For example, the COBRAcompounds of formula II include

Salts

[0083] The compounds of the invention are capable of forming bothpharmaceutically acceptable acid addition and/or base salts. Base saltsare formed with metals or amines, such as alkali and alkaline earthmetals or organic amines. Examples of metals used as cations are sodium,potassium, magnesium, calcium, and the like. Also included are heavymetal salts such as, for example, silver, zinc, cobalt, and cerium.Examples of suitable amines are N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamene,N-methylglucamine, and procaine.

[0084] Pharmaceutically acceptable acid addition salts are formed withorganic and inorganic acids. Examples of suitable acids for saltformation are hydrochloric, sulfuric, phosphoric, acetic, citric,oxalic, malonic, salicylic, malic, gluconic, fumaric, succinic,ascorbic, maleic, methanesulfonic, and the like. The salts are preparedby contacting the free base form with a sufficient amount of the desiredacid to produce either a mono or di, etc. salt in the conventionalmanner. The free base forms can be regenerated by treating the salt formwith a base. For example, dilute solutions of aqueous base can beutilized. Dilute aqueous sodium hydroxide, potassium carbonate, ammonia,and sodium bicarbonate solutions are suitable for this purpose. The freebase forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but the saltsare otherwise equivalent to their respective free base forms for thepurposes of the invention.

Depolymerization of Tubulin

[0085] The compounds of the invention bind to tubulin at the COBRAbinding pocket of tubulin. On binding of the tubulin binding compounds,tubulin is caused to depolymerize and/or inhibitition of tubulinassembly results. Suitable assays for the anti-tubulin acitivity of theinventive compounds are disclosed in the Examples below.

Treatment of Proliferative Disorders

[0086] The compounds of the invention are useful to inhibit celldivision and proliferation of non-cancerous cells. According to themethod of the inveniton, disorders associated with cell proliferationare treated by administration of the compounds and compositions of theinvention.

[0087] Such disorders include, for example, EBV-inducedlymphoproliferative disease and lymphoma; neointimal hypoplasia, forexample in patients with athlerosclerosis and patients undergoingballoon angioplasty; proliferative effects secondary to diabetes,including vascular proliferation and retinopathy; psoriasis; benigntumors, including angiomas, fiberomas, and myomas, histiocytosis,osteoporosis, mastocytosis, and myeleoproliferative disorders such aspolycytemiavera.

Tumor Treatment

[0088] The compounds of the invention are effective cytotoxic agents,for example, against tumor cells such as leukemia cells. In the methodsof the invention, the cytotoxic effects are achieved by contactingcells, such as tumor cells, with micromolar amounts of the inhibitorycompound.

[0089] The compounds of the invention can be used in methods of tumortreatment, for example, administering to a subject a compound of theinvention in order to achieve an inhibition of tumor cell tubulinassembly and/or depolymerization of tumor cell tubulin, inhibition oftumor cell growth, a killing of tumor cells, induced apoptosis, and/orincreased patient survival time.

[0090] The anti-cancer tubulin binding compounds of the invention aresuitable for use in mammals. As used herein, “mammals” means any classof higher vertebrates that nourish their young with milk secreted bymammary glands, including, for example, humans, rabbits, and monkeys.

Administration Methods

[0091] The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a mammalian host,including a human patient, in a variety of forms adapted to the chosenroute of administration. The compounds are preferably administered incombination with a pharmaceutically acceptable carrier, and can becombined with or conjugated to specific delivery agents, includingtargeting antibodies and/or cytokines.

[0092] The compounds can be administered by known techniques, such asorally, parentally (including subcutaneous injection, intravenous,intramuscular, intrasternal or infusion techniques), by inhalationspray, topically, by absorption through a mucous membrane, or rectally,in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants or vehicles.Pharmaceutical compositions of the invention can be in the form ofsuspensions or tablets suitable for oral administration, nasal sprays,creams, sterile injectable preparations, such as sterile injectableaqueous or oleagenous suspensions or suppositories.

[0093] For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents, andlubricants known in the art.

[0094] For administration by inhalation or aerosol, the compositions canbe prepared according to techniques well-known in the art ofpharmaceutical formulation. The compositions can be prepared assolutions in saline, using benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, or other solubilizing or dispersing agents known in theart.

[0095] For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

[0096] For rectal administration as suppositories, the compositions canbe prepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

[0097] Preferred administration routes include orally, parenterally, aswell as intravenous, intramuscular or subcutaneous routes.

[0098] More preferably, the compounds of the present invention areadministered parenterally, i.e., intravenously or intraperitoneally, byinfusion or injection. In one embodiment of the invention, the compoundscan be administered directly to a tumor by tumor injection. In anotherembodiment of the invention, the compounds can be administered usingsystemic delivery by intravenous injection.

[0099] Solutions or suspensions of the compounds can be prepared inwater, isotonic saline (PBS), and optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene, glycols, DNA, vegetable oils, triacetin and mixturesthereof. Under ordinary conditions of storage and use, thesepreparations can contain a preservative to prevent the growth ofmicroorganisms.

[0100] The pharmaceutical dosage form suitable for injection or infusionuse can include sterile, aqueous solutions, dispersions, or sterilepowders comprising an active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions. The final dosage form should be sterile, fluid andstable under the conditions of manufacture and storage. The liquidcarrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol such as glycerol,propylene glycol, or liquid polyethylene glycols, and the like,vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.The proper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size, in the caseof dispersion, or by the use of nontoxic surfactants. The prevention ofthe action of microorganisms can be accomplished by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the inclusion in thecomposition of agents delaying absorption such as, for example, aluminummonosterate hydrogels and gelatin.

[0101] Sterile injectable solutions are prepared by incorporating theconjugates in the required amount in the appropriate solvent withvarious other ingredients as enumerated above and, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying techniques, which yielda powder of the active ingredient plus any additional desired ingredientpresent in the previously sterile-filtered solutions.

Conjugation to a Targeting Moiety

[0102] The compound of the invention can be targeted for specificdelivery to the cells to be treated by conjugation of the compounds to atargeting moiety. Targeting moiety useful for conjugation to thecompounds of the invention include antibodies, cytokines, and receptorligands expressed on the cells to be treated.

[0103] The term “conjugate” refers to a complex formed with two or morecompounds.

[0104] The phrase “targeting moiety” refers to a compound which servesto deliver the compound of the invention to a specific site for thedesired activity. Targeting moieties include, for example, moleculeswhich specifically bind molecules present on a cell surface. Suchtargeting moieties useful in the invention include anti-cell surfaceantigen antibodies. Cytokines, including interleukins, factors such asepidermal growth factor (EGF), and the like, are also specific targetingmoieties known to bind cells expressing high levels of their receptors.

[0105] Particularly useful targeting moieties for targeting thecompounds of the invention to cells for therapeutic activity includethose ligands that bind antigens or receptors present on the tumor cellsto be treated. For example, antigens present on B-lineage cancer cells,such as CD19, can be targeted with anti-CD19 antibodies such as B43.Antibody fragments, including single chain fragments, can also be used.IL4 can also be used to target B-cells. Cancer cells expressing EGF orIGF receptors can be targeted with the binding ligand. Other suchligand-receptor binding pairs are known in the scientific literature forspecific cancers. Methods for producing conjugates of the compounds ofthe invention and the targeting moieties are known.

Useful Dose

[0106] When used in vivo to kill or inhibit the growth of tumor cells,the administered dose is that amount that can produce the desiredeffect, such as the amount sufficient to reduce or eliminate tumors.Appropriate amounts can be determined by those skilled in the art,extrapolating using known methods and relationships, from the in vitrodata provided in the Examples.

[0107] In general, the dose of the COBRA tubulin binding compoundseffective to achieve tumor cell apoptosis, reduction in tumors, andincreased survival time, is 1-100 mg/kg body weight/dose for a directtargeted administration. The effective dose to be administered will varywith conditions specific to each patient. In general, factors such asthe disease burden, tumor location (exposed or remote), host age,metabolism, sickness, prior exposure to drugs, and the like contributeto the expected effectiveness of a drug. One skilled in the art will usestandard procedures and patient analysis to calculate the appropriatedose, extrapolating from the data provided in the Examples.

[0108] In general, a dose which delivers about 1-100 mg/kg body weightis expected to be effective, although more or less can be useful.

[0109] In addition, the compositions of the invention can beadministered in combination with other anti-tumor therapies. In suchcombination therapy, the administered dose of the tubulin bindingcompounds can be less than for single drug therapy.

[0110] All publications, patents, and patent documents described hereinare incorporated by reference as if fully set forth. The inventiondescribed herein can be modified to include alternative embodiments. Allsuch obvious alternatives are within the spirit and scope of theinvention, as claimed below.

EXAMPLES

[0111] The invention can be further clarified by reference to thefollowing Examples, which serve to exemplify some of the embodiments,and not to limit the invention in any way.

Chemistry Methods

[0112] All chemicals were purchased from Aldrich (Milwaukee, Wis.) andwere used without further purification. Unless otherwise noted, eachreaction vessel was secured with a rubber septa, and the reaction wasperformed under nitrogen atmosphere. ¹H and ¹³C NMR spectra wereobtained on a Varian Mercury 300 instrument at ambient temperature inthe solvent specified. Melting points were determined using aFisher-Johns melting point apparatus and are uncorrected. FT-IR spectrawere recorded on a Nicolet Protege 460 spectrometer. GC/MS was obtainedon a HP 6890 GC System equipped with a HP 5973 Mass Selective Detecter.

Example 1 Reaction Schemes for Synthesis of AC-4

[0113] The tubulin depolymerizing COBRA compounds were quicklysynthesized in one step from commercially available starting materials.For example, AC-4 was prepared by reacting dodecylmagnesium chloride 1and 5-(hydroxymethyl)furfural 2 (Scheme 1). AC-4 was synthesized bymodification of the first generation of COBRA compounds, namely COBRA-1and COBRA-2 disclosed in U.S. Pat. No. 6,258,841 B1. Compared with thesynthesis of COBRA-1 and COBRA-2, the synthesis of AC-4 (Scheme 1) wasdramatically simplified by replacing the chiral THF moiety in COBRA-1and COBRA-2 with the achiral furan moiety in AC-4.

[0114] Dodecylmagnesium chloride (25 mL of 1M solution in ether) wasadded to the solution of 5-(hydroxymethyl)furfural (1.26 g, 10.0 mmol)in anhydrous ether (15 mL) at 0° C. The reaction mixture was stirred atroom temperature for 3 h and then quenched with saturated ammoniumchloride. The mixture was partitioned between ether (120 mL) and water(30 mL). The organic phase was washed with brine, dried over anhydrousMgSO₄, filtered and concentrated. Flash column chromatography furnishedcompound AC-4 as a white solid (2.58 g, 87%).

[0115] Melting point 56-57° C.; ¹H NMR (300 MHz, CDCl₃) δ 6.14 (d, J=3.5Hz, 1H), 6.09 (d, J=3.5 Hz, 1H, 4.55 (t, J=7.0 Hz, 1H), 4.46 (s, 2H),3.22 (bs, 1H), 3.06 (bs, 1H), 1.77 (m, 2H), 1.41−1.20 (m, 20H), 0.85 (t,J=6.5 Hz, 3H); ¹³C NMR (CDCl₃) δ 156.65, 153.10, 108.13, 106.33, 67.56,57.12, 35.28, 31.96, 29.73, 29.71, 29.67, 29.62, 29.48, 29.41, 25.70,22.75, 14.19; IR (neat) 3195, 2922, 2845, 1468, 1014 cm⁻¹.

Example 2 Synthesis and Characterization of AC-4 Analogs

[0116] A series of AC-4 analogs were synthesized.

[0117] Using Quest 205 Organic Synthesizer (Argonaut Technologies, SanCarlos, Calif.), compounds AC-6, AC-7, AC-10, AC-11, AC-12, AC-13,AC-15, AC-16 and AC-17 were synthesized in a parallel fashion to AC-4 byreacting dodecylmagnesium chloride 1 with the corresponding aldehydesunder the same conditions as shown in Scheme 1.

[0118] AC-14 was synthesized by refluxing 2-imidazolecarboxaldehyde 3and dodecylmagnesium chloride 1 in THF (Scheme 2) since 1 and 3 did notreact under the low temperature conditions used in Scheme 1.

[0119] AC-12 was synthesized by reacting 2,5-thiophene dicarboxaldehyde4 with dodecylmagnesium chloride 1 (Scheme 3).

AC-6

[0120] AC-6 was synthesized by following the method described for thesynthesis of AC-4.

[0121] Melting point: 40-41° C. ¹H NMR (300 MHz, CDCl₃) δ 7.35 (m, 1H),6.31 (m, 1H), 6.21 (m, 1H), 4.65 (m, 1H), 1.83 (m, 3H), 1.30 (m, 20H),0.86 (t, J=6.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 156.73, 141.73,110.02, 105.69, 67.85, 35.61, 31.99, 29.74, 29.72, 29.65, 29.60, 29.47,29.43, 25.62, 22.78, 14.24; IR (neat) 3350, 2916, 2848, 1457, 725 cm⁻¹.

AC-7

[0122] AC-7 was an orange waxy liquid; synthesized by following themethod described for the synthesis of AC-4.

[0123]¹H NMR (300 MHz, CDCl₃) δ 7.83 (dd, J=8.0, 1.5 Hz, 1H), 7.41 (dd,J=8.0, 1.5 Hz, 1H), 7.29 (ddd, J=8.0, 8.0, 1.5 Hz, 1H), 7.16 (ddd,J=8.0, 8.0, 1.5 Hz, 1H), 7.05 (d, J=3.0 Hz, 1H), 6.35 (d, J=3.0 Hz, 1H),4.71 (m, 1H), 2.03 (d, J=3.5 Hz, 1H), 1.89 (m, 2H), 1.49−1.24 (m, 20H),0.87 (t, J=6.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 156.42 149.26, 130.56,129.87, 128.98, 127.83, 127.66, 126.69, 111.46, 107.87, 67.92, 35.70,31.98, 29.74, 29.71, 29.65, 29.60, 29.48, 29.43, 25.61, 22.77, 14.23; IR(neat) 3346, 2924, 2854, 1471, 1022, 754 cm⁻¹.

AC-10

[0124] AC-10 was a brown liquid, synthesized by following the methoddescribed for the synthesis of AC-4.

[0125]¹H NMR (300 MHz, CDCl₃) δ 7.24 (m, 1H), 6.95 (m, 2H), 4.90 (t,J=6.5 Hz, 1H), 2.01 (s, 1H), 1.82 (m, 2H), 1.43 (m,1H), 1.23 (m, 19H),0.86 (t, J=6.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 148.81, 126.45,124.34, 123.57, 70.36, 39.36, 31.98, 29.73, 29.70, 29.64, 29.59, 29.45,29.42, 25.86, 22.77, 14.22; IR (neat) 3354, 2924, 2854, 2361, 1466, 696cm⁻¹.

AC-11

[0126] AC-11 was a yellowish wax, synthesized by following the methoddescribed for the synthesis of AC-4.

[0127]¹H NMR (300 MHz, CDCl₃) δ 6.87 (d, J=4.0 Hz, 1H), 6.68 (d, J=4.0Hz, 1H), 4.79 (t, J=6.5 Hz, 1H), 2.01 (bs, 1H), 1.77(m, 2H), 1.45−1.16(m, 20H), 0.86 (t, J=6.5, 3H); ¹³C NMR(75 MHz, CDCl₃) δ 150.45, 129.18,123.84, 111.22, 70.59, 39.13, 31.99, 29.73, 29.71, 29.68, 29.57, 29.43,29.40, 25.70, 22.78, 14.24; IR (neat) 3334, 2924, 2852, 1466, 1444, 968,795 cm⁻¹.

AC-12

[0128] Using the ratio of 2,5-thiophene dicarboxaldehyde 4:dodecylmagnesium chloride 1=1:1, AC-12 was synthesized by following theconditions described in the synthesis of AC-4.

[0129]¹H NMR (300 MHz, CDCl₃) δ 9.62 (s, 1H); 7.01 (d, J=4.0 Hz, 1H);6.61 (t, J=4.0 Hz, 1H); 4.44 (m, 1H), 1.55 (m, 2H), 1.37−1.20 (m, 21H);0.93 (t, J=6.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 182.80, 160.09,142.17, 136.37, 124.30, 70.55, 39.41, 31.98, 29.71, 29.70, 29.68, 29.61,29.55, 29.41, 29.39, 25.50, 22.77, 14.22; IR (neat) 3417, 2924, 2854,1668, 1460 cm⁻¹.

AC-13

[0130] AC-13 was synthesized by following the method described for thesynthesis of AC-4.

[0131] Melting point: 60-61° C.; ¹H NMR (300 MHz, CDCl₃) δ 7.68 (d,J=3.5 Hz, 1H), 7.26 (d, J=3.5 Hz, 1H), 4.97 (m, 1H), 3.23 (d, J=4.5,1H), 1.98-1.77 (m, 2H), 1.50−1.37 (m, 2H), 1.23 (bs, 18H), 0.86 (t,J=6.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 175.24, 141.97, 118.71, 71.91,38.38, 31.98, 29.73, 29.70, 29.63, 29.56, 29.45, 29.42, 25.25, 22.77,14.22; IR (neat) 3236, 2914, 2848 1470, 743 cm⁻¹.

AC-14

[0132] AC-14 was synthesized by following the method described for thesynthesis of AC-4).

[0133] Melting point: 97-98° C.; ¹H NMR (300 MHz, CD₃OD) δ 6.98 (s, 2H),4.74 (t, J=6.5, 1H), 1.85 (m, 2H), 1.31 (bs, 20H), 0.93 (t, J=6.5 Hz,3H); ¹³C NMR (75 MHz, CD₃OD) δ 153.02, 122.97, 69.86, 38.72, 33.98,31.69, 31.65, 31.58, 31.38, 27.22, 24.65, 15.37; IR (neat) 3163, 3078,2916, 2848, 1468 cm⁻¹.

AC-15

[0134] AC-15 was synthesized by following the method described for thesynthesis of AC-4.

[0135] Melting point: 0.300° C., ¹H NMR (300 MHz, CD₃OD) δ 7.49 (s, 1H),5.04 (bs, 2H), 4.70 (t J=7.0 Hz, 1H), 2.24 (s, 3H), 1.86 (m, 2H), 1.31(m, 20H), 0.93 (t, J=6.5 Hz, 3H); ¹³C NMR (75 MHz, CD₃OD) δ 135.19,68.01, 38.74, 34.01, 31.76, 31.73, 31.70, 31.66, 31.65, 31.51, 31.43,27.85, 24.69, 15.45, 11.74; IR (neat) cm⁻¹.

AC-16

[0136] AC-16 was synthesized by following the method described for thesynthesis of AC-4.

[0137] Melting point: 50-52° C.; ¹H NMR (300 MHz, CD₃OD) δ 7.53 (dd,J=7.5, 7.5 Hz, 1H), 7.01 (d, J=7.5 Hz, 1H), 7.00 (d, J=7.5 Hz), 4.66(dd, J=4.0, 8.0 Hz, 1H), 4.46 (bs, 1H), 2.52 (s,3H), 1.76 (m, 1H), 1.63(m, 1H), 1.40 (m, 2H), 1.22 (m,18H), 0.86 (t, J=6.5 Hz, 3H); ¹³C NMR (75MHz, CD₃OD) δ 161.12, 156.73, 136.71, 121.48, 117.04, 72.30, 38.80,31.99, 29.73, 29.69, 29.66, 29.43, 25.41, 24.34, 22.78, 14.24; IR (neat)3244, 2922, 2850, 2361 cm⁻¹.

AC-17

[0138] AC-17 was synthesized by following the method described for thesynthesis of AC-4.

[0139] Melting point: 49-50° C.; ¹H NMR (300 MHz, CD₃OD) δ 8.50 (d,J=5.0 Hz, 1H), 7.64 (ddd, J=7.5, 7.5, 1.5 Hz, 1H), 7.23 (d, J=7.8 Hz,1H), 7.16 (ddd, J=7.2, 5.0, 0.9 Hz, 1H), 4.70 (dd, J=4.5, 7.5 Hz, 1H),4.20 (bs, 1H), 1.77 (m, 1H), 1.66 (m, 1H), 1.38 (m, 2H), 1.22 (bs, 18H),0.85 (t, J=6.5 Hz, 3H); ¹³C NMR (75 MHz, CD₃OD) δ 162.17, 147.98,136.44, 122.06, 120.20, 72.77, 38.69, 31.97, 29.72, 29.70, 29.68, 29.66,29.63, 29.41, 25.35, 22.76, 14.21; IR (neat) 3385, 2924, 2854, 1595,1468, 1047 cm⁻¹.

Example 3 Anti-Cancer Activity of COBRA Compounds as Measured by MTTAssays

[0140] The following human cancer cell lines were used in the presentstudy: human leukemic cell lines Molt-3 and NALM-6. These cell lineswere obtained from American Type Culture Collection (Manassas, Va.) andmaintained as continuous cell lines in Dulbecco's modified Eagles'smedium supplemented with 10% fetal bovine serum and antibiotics.

[0141] The antiproliferative activity of the COBRA compounds wasexamined against a panel of 2 different human tumor cell lines usingstandard MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assays (Narla, R. K., Liu, X. P., Klis, D. and Uckun, F. M.,Clin. Cancer Res., 1998, 4, 2463), (Boehringer Mannheim Corp.,Indianapolis, Ind.). Briefly, exponentially growing brain tumor cellswere seeded into a 96-well plate at a density of 2.5×10⁴ cells/well andincubated for 36 hours at 37° C. prior to drug exposure. On the day oftreatment, culture medium was carefully aspirated from the wells andreplaced with fresh medium containing the test compounds atconcentrations ranging from 1.0 to 250 μM. Triplicate wells were usedfor each treatment. The cells were incubated with the various compoundsfor 24-36 hours at 37° C. in a humidified 5% CO₂ atmosphere. To eachwell, 10 μl of MTT (0.5 mg/ml final concentration) was added and theplates were incubated at 37° C. for 4 hours to allow MTT to formformazan crystals by reacting with metabolically active cells. Theformazan crystals were solubilized overnight at 37° C. in a solutioncontaining 10% SDS in 0.01 M HCl. The absorbence of each well wasmeasured in a microplate reader (Labsystems) at 540 nm and a referencewavelength of 690 nm. To translate the OD ₅₄₀ values into the number oflive cells in each well, the OD ₅₄₀ values were compared to those onstandard OD ₅₄₀—versus—cell number curves generated for each cell line.The percent survival was calculated using the formula: survival=Livecell number[test]×100/Live cell number [control].

[0142] IC₅₀ values were calculated by non-linear regression analysisusing an Graphpad Prism software version 2.0 (Graphpad Software, Inc.,San Diego, Calif.). TABLE 1 Anti-Cancer Activity COBRA Compound MOLT-3Lymphoma NALM-6 Leukemia Compound IC₅₀ (μM) IC₅₀ (μM) AC-6  >250 229AC-7  >250 >250 AC-10 125 204 AC-11 2 4 AC-12 33 6 AC-13 4 >250 AC-14 583 AC-15 9 25 AC-16 >250 236 AC-17 246 221

[0143] All publications, patents, and patent documents described hereinare incorporated by reference as if fully set forth. The inventiondescribed herein can be modified to include alternative embodiments. Allsuch obvious alternatives are within the spirit and scope of theinvention, as claimed below.

We claim:
 1. A compound of formula I:

wherein X is O, S, or NH; R is a saturated or unsaturated (C₇ to C₁₅)hydrocarbon chain; R¹ is hydrogen, halogen, OH, (C₁ to C₆) alkoxy, (C₁to C₆) acyl, (C₁ to C₆) ester, or (C₁ to C₆) carboxylic acid; Y is OH,SH, CN, halogen, or (C₁ to C₆) alkoxy; and pharmaceutically acceptablesalts thereof.
 2. The compound of claim 1, wherein R is a C₁₂ alkyl or aC₁₂ alkylene.
 3. The compound of claim 1, wherein X is S or O.
 4. Thecompound of claim 1, wherein R is C₁₂H₂₅; R¹ is hydrogen, bromine, CHO,or COOH; Y is OH; and X is oxygen or sulfur.
 5. The compound of claim 1,having the formula


6. The compound of claim 1, having the formula


7. The compound of claim 1, having the formula


8. A compound of formula II:

wherein X is NH, O, or S; R is a saturated or unsaturated (C₇ to C₁₅)hydrocarbon chain; R² is hydrogen, OH, (C₁ to C₆) alkoxy, (C₁ to C₆)alkyl, (C₁ to C₆)alkenyl, (C₁ to C₆) alkynyl, (C₃ to C₇) cycloalkyl,aryl, heteroaryl, halogen, (C₁ to C₆) acyl, (C₁ to C₆) ester, or (C₁ toC₆) carboxylic acid; Y is OH, SH, CN, halogen, or (C₁ to C₆) alkoxy; andpharmaceutically acceptable salts thereof.
 9. The compound of claim 8,wherein R is is a C₁₂ alkyl or a C₁₂ alkylene.
 10. The compound of claim8, wherein R is C₁₂H₂₅; Y is OH; X is S or NH; and R² is hydrogen or a(C₁ to C₆) alkyl.
 11. The compound of claim 8, having the formula


12. The compound of claim 8, having the formula


13. A compound of formula III:

wherein X is NH, O, or S; R is a saturated or unsaturated (C₇ to C₁₅)hydrocarbon chain; R² is hydrogen, OH, (C₁ to C₆) alkoxy, (C₁ to C₆)alkyl, (C₁ to C₆)alkenyl, (C₁ to C₆) alkynyl, (C₃ to C₇) cycloalkyl,aryl, heteroaryl, halogen, (C₁ to C₆) acyl, (C₁ to C₆) ester, or (C₁ toC₆) carboxylic acid; Y is OH, SH, CN, halo, or (C₁ to C₆) alkoxy; andpharmaceutically acceptable salts thereof.
 14. The compound of claim 13,wherein R is a C₁₂ alkyl or a C₁₂ alkylene.
 15. The compound of claim13, wherein R is C₁₂H₂₅; Y is OH; X is S or NH; and R² is hydrogen or a(C₁ to C₆) alkyl.
 16. The compound of claim 13, having the formula


17. A composition comprising the compound of claim 1 and apharmaceutically acceptable carrier or diluent.
 18. The composition ofclaim 17, wherein the compound is


19. The composition of claim 17, wherein the compound is


20. The composition of claim 17, wherein the compound is


21. A composition comprising the compound of claim 8 and apharmaceutically acceptable carrier or diluent.
 22. The composition ofclaim 21, wherein the compound is


23. The composition of claim 21, wherein the compound is


24. A composition comprising the compound of claim 13 and apharmaceutically acceptable carrier or diluent.
 25. The composition ofclaim 24, wherein the compound is


26. A method for inhibiting the proliferation of tumor cells comprisingadministering to said tumor cells an inhibitory amount of the compoundof claim
 1. 27. A method for inhibiting the proliferation of tumor cellscomprising administering to said tumor cells an inhibitory amount of thecompound of claim
 8. 28. A method for inhibiting the proliferation oftumor cells comprising administering to said tumor cells an inhibitoryamount of the compound of claim
 13. 29. A method for treating cancer ina patient comprising administering to said patient a therapeuticallyeffective amount of a compound of claim
 1. 30. The method of claim 29,wherein the cancer is leukemia.
 31. A method for treating cancer in apatient comprising administering to said patient a therapeuticallyeffective amount of a compound of claim
 8. 32. The method of claim 31,wherein the cancer is leukemia.
 33. A method for treating cancer in apatient comprising administering to said patient a therapeuticallyeffective amount of a compound of claim
 13. 34. The method of claim 33,wherein the cancer is leukemia.
 35. A method for inducing cytotoxicityin a cell comprising administering to said cell a cytotoxic dose of acompound of claim
 1. 36. The method of claim 35, wherein the compound is


37. The method of claim 35, wherein the compound is


38. The method of claim 35, wherein the compound is


39. A method for inducing cytotoxicity in a cell comprisingadministering to said cell a cytotoxic dose of a compound of claim 8.40. The method of claim 39, wherein the compound is


41. The method of claim 39, wherein the compound is


42. A method for inducing cytotoxicity in a cell comprisingadministering to said cell a cytotoxic dose of a compound of claim 13.43. The method of claim 42, wherein the compound is